SYNAPSE PROJECT [unreadable] [unreadable] The post synaptic density (PSD) at excitatory glutamatergic synapses is a complex molecular machine which appears to be a key site of information storage. The PSDs in intact neurons, however, change size rapidly and reversibly during activity. Immunolabeling shows that CaMKII is a major component of the added mass involving addition of up to 100 CaMKII holoenzymes. Comparison of PSD dynamics in the cerebellum and forebrain indicate a correlation between cellular alpha-CaMKII abundance and degree of PSD thickening. We have now shown that at synapses in intact cerebral cortex CaMKII is added to the PSD after activity. Both the thickening and addition of CaMKII persists in LTP, suggesting that the induced association with the PSD is a key step leading to LTP. In order to explore the detailed molecular organization of the PSD, we have developed a method to freeze-substitute hippocampal cultures and then examine them by tomography of thin sections. Tomography reveals that the core of the PSD is a large array of vertically oriented PSD-95 filaments. We also identify AMPA and NMDA receptors, which are both contacted by the PSD-95 filaments. There are two major types of filaments oriented parallel to the post synaptic membrane. One type contacts the vertical filaments that also contact the NMDA receptors, while the other type contacts all vertical filaments. Most receptors in the spine membrane outside of the PSD are not contacted by filaments. These finding solve the core molecular structure of the PSD. Ongoing complementary studies with isolated PSDs, using rotary shadowing, have mapped the distributions of PSD-specific scaffolding molecules, including PSD-93 and SAP-97 within the PSD. We affinity purify PSDs from other components of the PSD fraction, thereby allowing independent measurement of CaMKII content, as well as proteomic analysis by mass spectroscopy (with S. Markey). This study was the first proteomic analysis of purified PSDs, thereby eliminating the contributions from abundant contaminating cytoskeletal elements and other proteins in the PSD fraction. Comparison of mass spectrometric data for the conventional and affinity-purified PSD fractions reveals enrichment following affinity purification of specialized scaffolding molecules and glutamate receptors, especially of the AMPA type, accompanied by a notable decrease in certain cytoskeletal and presynaptic proteins formerly regarded as core components of the PSD.